Pancreatic cancer is almost invariably associated with mutations in the KRAS gene, most commonly KRAS G12D , that result in a dominant-active form of the KRAS GTPase. However, how KRAS mutations promote pancreatic carcinogenesis is not fully understood, and whether oncogenic KRAS is required for the maintenance of pancreatic cancer has not been established. To address these questions, we generated two mouse models of pancreatic tumorigenesis: mice transgenic for inducible Kras G12D , which allows for inducible, pancreas-specific, and reversible expression of the oncogenic Kras G12D , with or without inactivation of one allele of the tumor suppressor gene p53. Here, we report that, early in tumorigenesis, induction of oncogenic Kras G12D reversibly altered normal epithelial differentiation following tissue damage, leading to precancerous lesions. Inactivation of Kras G12D in established precursor lesions and during progression to cancer led to regression of the lesions, indicating that Kras G12D was required for tumor cell survival. Strikingly, during all stages of carcinogenesis, Kras G12D upregulated Hedgehog signaling, inflammatory pathways, and several pathways known to mediate paracrine interactions between epithelial cells and their surrounding microenvironment, thus promoting formation and maintenance of the fibroinflammatory stroma that plays a pivotal role in pancreatic cancer. Our data establish that epithelial Kras G12D influences multiple cell types to drive pancreatic tumorigenesis and is essential for tumor maintenance. They also strongly support the notion that inhibiting Kras G12D , or its downstream effectors, could provide a new approach for the treatment of pancreatic cancer.

Chronic obstructive pulmonary disease (COPD) is increasingly being recognized as a highly heterogeneous disorder, composed of varying pathobiology. Accurate detection of COPD subtypes by image biomarkers are urgently needed to enable individualized treatment thus improving patient outcome. We adapted the Parametric Response Map (PRM), a voxel-wise image analysis technique, for assessing COPD phenotype. We analyzed whole lung CT scans of 194 COPD individuals acquired at inspiration and expiration from the COPDGene Study. PRM identified the extent of functional small airways disease (fSAD) and emphysema as well as provided CT-based evidence that supports the concept that fSAD precedes emphysema with increasing COPD severity. PRM is a versatile imaging biomarker capable of diagnosing disease extent and phenotype, while providing detailed spatial information of disease distribution and location. PRMs ability to differentiate between specific COPD phenotypes will allow for more accurate diagnosis of individual patients complementing standard clinical techniques.

The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.

Exposure to short-wavelength UV light (UVC) strongly induces p53 expression. In human RKO colorectal carcinoma cells, this increase was not due to elevated p53 mRNA abundance, cytoplasmic export of p53 mRNA, or UVC-triggered stabilization of the p53 protein. Instead, p53 translation was potently enhanced after UVC irradiation. The 3 UTR of p53 was found to be a target of the RNAbinding protein HuR in a UVC-dependent manner in vitro and in vivo. HuR-overexpressing RKO cells displayed elevated p53 levels, whereas cells expressing reduced HuR showed markedly diminished p53 abundance and p53 translation. Our results demonstrate a role for HuR in binding to the p53 mRNA and enhancing its translation.UV light ͉ embryonic lethal abnormal vision

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The RNA-binding protein TIA-1 (T-cell intracellular antigen 1) functions as a posttranscriptional regulator of gene expression and aggregates to form stress granules following cellular damage. TIA-1 was previously shown to bind mRNAs encoding tumor necrosis factor alpha (TNF-␣) and cyclooxygenase 2 (COX-2), but TIA-1 target mRNAs have not been systematically identified. Here, immunoprecipitation (IP) of TIA-1-RNA complexes, followed by microarray-based identification and computational analysis of bound transcripts, was used to elucidate a common motif present among TIA-1 target mRNAs. The predicted TIA-1 motif was a U-rich, 30-to 37-nucleotide (nt)-long bipartite element forming loops of variable size and a bent stem. The TIA-1 motif was found in the TNF-␣ and COX-2 mRNAs and in 3,019 additional UniGene transcripts (ϳ3% of the UniGene database), localizing preferentially to the 3 untranslated region. The interactions between TIA-1 and target transcripts were validated by IP of endogenous mRNAs, followed by reverse transcription and PCR-mediated detection, and by pulldown of biotinylated RNAs, followed by Western blotting. Further studies using RNA interference revealed that TIA-1 repressed the translation of bound mRNAs. In summary, we report a signature motif present in mRNAs that associate with TIA-1 and provide support to the notion that TIA-1 represses the translation of target transcripts.

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Here, we present evidence that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR was specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was linked to its decreased association with 14-3-3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14-3-3 and hindering its post-transcriptional function and anti-apoptotic influence.[Keywords: RNA-binding protein; nucleocytoplasmic shuttling; 14-3-3 proteins; post-transcriptional gene regulation; cell division cycle; elav] Supplemental material is available at http://www.genesdev.org.

The ability of the vertebrate X-linked inhibitor of apoptosis (XIAP) protein to directly suppress apoptotic cell death pathways has been the subject of much research. Studies of this broadly expressed protein have largely focused on the unique interactions between XIAP and caspases -proteases that conduct and participate in the ordered disassembly of the cell during apoptosis. However, relatively less attention has been given to the RING domain of XIAP, which functions as an E3 ligase to catalyze the ubiquitination of substrate proteins. Here, we discuss the evidence implicating the RING domain of XIAP in the ubiquitinmediated regulation of three, somewhat arbitrarily divided, categories of substrate: XIAP itself, XIAP-interacting proteins involved in apoptosis, and other targets whose physiological roles likely extend beyond cell death. Collectively, these multiple activities of XIAP show that this enigmatic protein participates in a range of cellular activities beyond apoptotic suppression. Despite the immeasurable value to the biomedical research community of readily accessible, easily searchable DNA sequence databases, a major challenge created by this explosion in the identification of new genes is to understand the physiological functions of their products. The X-linked Inhibitor of apoptosis (XIAP) is a good example of this. Originally identified by its homology to the Iap genes contained within the genomes of baculoviruses, 1-3 which themselves were discovered in genetic screens for suppressors of cell death 4,5 , XIAP has been shown to participate in a range of cellular activities that include, but are not limited to, apoptotic regulation. 6 This has created a challenge in understanding how XIAP can be such a versatile, multifunctional protein, and how these apparently unrelated cellular roles might be reconciled.Cellular IAP (c-IAP) proteins contain between one and three baculovirus IAP repeats (BIRs), B70-residue zinc-binding domains that are named from their original discovery in baculoviruses and that resemble a classical zinc finger configuration. 7-9 XIAP contains three BIRs (Figure 1), which, together with flanking residues, can bind directly to caspases-3, -7 and -9, thereby inhibiting their proteolytic activity. The structural features of these interactions, together with the mechanisms by which XIAP can inhibit the enzymatic activities of these caspases, have been studied in detail, 11 yet despite the abundance of biochemical data supporting an extremely high affinity of XIAP for caspases, there are surprisingly few published reports describing endogenous XIAP/caspase interactions. 12 Nevertheless, based on these structural studies XIAP is considered to be the only mammalian IAP protein that can function as a direct, competitive inhibitor of the enzymatic activity of caspases through binding to their catalytically active sites. 11 Although the presence of a BIR is often considered as the defining feature of IAPs, many c-IAPs contain a second class of domain, which is also found in the baculovi...